bioRxiv preprint doi: https://doi.org/10.1101/2020.05.18.097154; this version posted May 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. SDE2 Integrates into the TIMELESS-TIPIN Complex to Protect Stalled Replication Forks Julie Rageul1,6, Jennifer J. Park1,6, Ping Ping Zeng1, Eun-A Lee2, Jihyeon Yang2, Sunyoung Hwang2, Natalie Lo1, Alexandra S. Weinheimer3, Orlando D. Schärer2,4, Jung-Eun Yeo2*, and Hyungjin Kim1,5* 1Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794, USA 2Center for Genomic Integrity, Institute for Basic Science, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea 3Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794, USA 4Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea 5Stony Brook Cancer Center, Renaissance School of Medicine at Stony Brook University, Stony Brook, New York 11794, USA 6These authors contributed equally * Correspondence: Hyungjin Kim, Ph.D. Department of Pharmacological Sciences Renaissance School of Medicine at Stony Brook University Basic Sciences Tower 8-125 100 Nicolls Rd., Stony Brook, NY 11794, USA [email protected] Jung-Eun Yeo, Ph.D. Center for Genomic Integrity, Institute for Basic Science (IBS), UNIST-gil 50, Ulsan 44919, Republic of Korea [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.18.097154; this version posted May 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances TIM stability and its localization to replication forks, thereby aiding the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization. Keywords: Genome stability, DNA replication stress, Replication fork protection, SDE2, Fork protection complex, TIMELESS 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.18.097154; this version posted May 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. INTRODUCTION DNA replication is one of the most fundamental biological processes for the survival of an organism. Eukaryotic DNA replication is coordinated by specialized replisome machinery, including the Cdc45-MCM-GINS (CMG) helicase complex that unwinds the parental duplex DNA, and replicative polymerases that synthesize the daughter strand1,2. Additionally, proliferating cell nuclear antigen (PCNA) is a DNA clamp that acts not only as a processivity factor to guide DNA polymerases, but also as a scaffold to coordinate the replication stress response that protects against DNA damage3. The DNA replication fork inevitably exposes single-stranded DNA (ssDNA) and is prone to damage that arises from various intrinsic and extrinsic barriers against fork progression4. Improper control of accumulated DNA replication stress results in the stalling and subsequent instability of replication forks, rendering DNA susceptible to nucleolytic degradation and breakage, triggering genome instability and tumor development5. Due to their hyper-proliferative nature, cancer cells are often selected through loss of mechanisms to control DNA replication and exhibit elevated levels of replication stress6. Thus, exacerbating DNA replication stress in cancer cells has emerged as a new strategy to specifically kill cancer cells. Cells have evolved sophisticated genome surveillance mechanisms to protect stalled replication forks and preserve fork integrity. The DNA replication stress response pathway, primarily driven by the intra-S checkpoint that activates ATR-dependent CHK1 phosphorylation, acts to inhibit cell cycle progression, stimulate DNA repair, modulate origin firing, and assist in the restart of stalled replication forks7. RPA-coated ssDNA, generated by the uncoupling of the CMG helicase and replicative polymerase activities, acts as a platform to recruit the ATR- binding partner ATRIP and stimulate ATR kinase activity through TOPBP1 at the 5´ ssDNA- double-stranded DNA (dsDNA) junction and also RPA-mediated ETAA1 recruitment, leading to CHK1 activation8-12. Additionally, emerging evidence suggests that fork reversal plays a key role in protecting stressed replication forks13. This process involves regression of a stalled fork to 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.18.097154; this version posted May 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. form a four-way junction by the action of the RAD51 recombinase and several SWI/SNF family translocases such as SMARCAL1, ZRANB3, and HLTF14,15. This unique transaction protects stressed forks from degradation and acts as an intermediate for the repair and restart of stalled forks16-18. The tumor suppressor proteins BRCA1/2 and Fanconi anemia (FA) protein FANCD2 are required for the protection of reversed forks by stabilizing the RAD51 filaments and preventing the regressed arm from nucleolytic degradation19. Many regulatory proteins fine-tune the steps of processing or protecting reversed forks, and deregulated fork remodeling has been associated with fork collapse, genome instability, and sensitivity or resistance to chemotherapy20,21. The fork protection complex (FPC), composed of TIMELESS (TIM) and TIPIN (Swi1 and Swi3 in S. pombe, and Tof1 and Csm3 in S. cerevisiae), and the ancillary proteins AND-1 and CLASPIN (CLSPN) stabilizes the replisome to ensure unperturbed fork progression22-24. The FPC acts as a scaffold to link the movements of the CMG helicase and polymerase, preventing the uncoupling of their activities and ensuring efficient replisome progression25,26. Additionally, the FPC promotes the ATR-CHK1 checkpoint signaling under replication stress by stimulating the association of TIM-TIPIN and CLSPN to RPA-ssDNA at stalled forks, thereby facilitating CLSPN-mediated CHK1 phosphorylation by ATR27,28. TIM and TIPIN form an obligate heterodimer and have no known enzymatic function, suggesting that they play a structural role in supporting replisome integrity29. Loss of the FPC leads to defects in DNA replication and genome instability, indicating that maintaining structural integrity of the replisome is critical for preserving fork stability30-32. TIM is upregulated in a variety of cancers, implying that enhanced FPC activity may alleviate replication stress arising in tumors through oncogene activation33. However, the regulatory mechanism through which the FPC is controlled at active and stalled replication forks remains unclear. We previously identified human SDE2 as a PCNA-associated protein required for counteracting replication-associated DNA damage34. SDE2 contains a ubiquitin-like (UBL) motif 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.18.097154; this version posted May 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. at its N-terminus that is cleaved off in a PCNA-dependent manner to release a C-terminal (Ct) SDE2Ct fragment (Figure 1A). SDE2Ct contains a conserved SDE2 domain of unknown function at its N-terminus, while its C-terminal SAP domain mediates the association of SDE2Ct with chromatin. We further showed that chromatin-associated degradation of SDE2Ct by Arg/N-end rule-p97 ATPase proteolytic pathway is necessary for propagating the signaling of the DNA replication stress response at RPA-coated stalled forks in response to UVC damage35. These findings indicate that the dynamic control of SDE2 protein levels may modulate protein complexes and their activities at stressed forks. Although a role of SDE2 in DNA replication and stalled fork recovery has been shown, the mechanism by which this occurs is unknown34. Here, we demonstrate that SDE2 directly interacts with the FPC component TIM and promotes TIM stability and its localization at replication forks. Consequently, loss of SDE2 or TIM compromises the integrity of the FPC, leading to defects in fork progression, stalled fork recovery, and checkpoint activation. Notably, SDE2 cooperates with TIM in protecting reversed forks from unrestricted nuclease activity, a role not previously associated